DNA supercoiling is involved with virtually all DNA-related biochemical processes, therefore physical chemical and structural properties of supercoiled DNA have been important areas of DNA biophysics over last two decades. Recent experimental studies and advanced theoretical analyses suggest that DNA dynamics rather than static DNA structure plays a critical role in the control of gene expression, DNA replication, recombination, and repair. However, progress in experimental studies in this important area of DNA biophysics has been very low due to the lack of appropriate experimental techniques. Recent advances in single molecule studies, allowing the observation of structural dynamics of individual molecules, showed that the single molecule approaches could provide unique information on the structure and dynamics of biological molecules. Recent studies showed that local and global DNA conformations are in a dynamic equilibrium. Competing transitions between different local structures accompanied by dynamic changes of global DNA conformations may have a profound role in various DNA functions. This proposal utilizing single molecule AFM and fluorescence microscopy tests a number of critical hypotheses and provides a realistic means for breaking new ground in this undeveloped area of molecular biology. (1) The hypothesis that the formation of cruciforms and H-DNA can limit global DNA mobility affecting the juxtaposition of distantly separated DNA regions will be tested. (2) The hypothesis that conversion of one local structure into another is an alternative molecular switch mechanism for the global DNA structure and dynamics (3) The mechanism of binding of the restriction enzyme SfiI involving the interaction of distant DNA regions will be analyzed and the hypothesis that the formation of local structures may regulate the enzyme activity will be tested. In addition to the hypotheses testing, novel DNA immobilization and labeling techniques for DNA dynamics studies will be developed. Altogether, the proposed studies are the first attempt to look at the dynamics of DNA at various levels providing spatial and temporal information about the fundamental and functional properties of DNA. Advances in sample preparation techniques combined with corresponding instrument development will provide structural biologists pursuing single molecule approaches with novel tools for applications, ramifications considerably wider than the objectives outlined in this proposal.